Regeneration and neogenesis of retinal visual cell-expressing Otx2 protein

ABSTRACT

The present invention provides a medicine, comprising (a) an Otx2 protein or its partial peptide, or a salt thereof, or (b) a DNA or an RNA encoding an Otx2 protein or its partial peptide. The present medicine is useful as an agent for preventing, treating or suppressing progression of a retinal disease including retinal degeneration. In addition, the present medicine is useful, for example, as an agent for inducing differentiation from a retinal stem cell into a retinal photoreceptor cell, in the transplantation of a cell into the retina of patients suffering from retinal diseases.

This application is a U.S. national stage of International ApplicationNo. PCT/JP2004/001023 filed Feb. 2, 2004.

TECHNICAL FIELD

The present invention relates to an agent for inducing differentiationinto retinal photoreceptor cells, an agent forpreventing/treating/suppressing progression of retinal diseases, and adiagnostic agent for retinal diseases, which contain an Otx2 protein ora gene encoding an Otx2 protein.

BACKGROUND ART

Retinal photoreceptor cells are only one photosensor in a mammal, andhas previously been intensively studied physiologically, biochemically,anatomically and clinically (JP-A-2002-325571). However, mechanism ofdevelopment and differentiation into retinal photoreceptor cells hasbeen unknown at all. As a causative locus for human genetic retinaldegeneration which is a disease resulting from abnormalities of retinalphotoreceptor cells, at least 145 loci are known, but an establishedmethod of treating the disease has not been present yet, and patientsare suffering from severe eyesight disorder. Therefore, elucidation ofcauses, and establishment of therapeutic method for retinal degenerationleading to loss of eyesight or severe eyesight disorder have beendesired. In addition, since degeneration or dyscrasia of retinalphotoreceptor cells is seen in many retinal diseases such as not onlyretinitis pigmentosa, but also diabetic retinopathy and maculardegeneration, it is very important to elucidate molecular mechanism ofdevelopment and differentiation of retinal photoreceptor cells, in orderto enable degeneration or neogenesis of retinal photoreceptor cells,

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide an agent forpreventing, treating or suppressing progression of retinal diseasesincluding retinal degeneration. Another object of the present inventionis to provide a method for screening a compound useful as medicines. Afurther object of the present invention is to provide an agent of, or amethod of diagnosing retinal diseases. A still further object of thepresent invention is to provide a method for inducing differentiation,or an agent for inducing differentiation into retinal photoreceptorcells which are suitable for transplantation in the retina of patientssuffering from retinal diseases.

The present inventors have paid their attention to an Otx2 proteinbelonging to the same gene family as that of a transcription factor Crxwhich has previously been analyzed as a key of differentiation intoretinal photoreceptor cells, and analyzed the role of an Otx2 protein indetermining the fate of a retinal photoreceptor cell. As a result ofanalysis at a level of a mouse living body, the present inventors havefound that an Otx2 protein is very important in determining the fate ofa retinal photoreceptor cell. That is, when an Otx2 gene wasincorporated into a virus vector to infect a mouse undifferentiatedretinal stem cell with the recombinant vector, and to express an Otx2protein in a mouse undifferentiated retinal stem cell, most ofundifferentiated retinal stem cells were differentiated into retinalphotoreceptor cells. Based on such finding, the present inventors havefurther studied to complete the present invention.

That is, the present invention relates to:

(1) an agent for preventing, treating or suppressing progression of aretinal disease, which comprises an Otx2 protein or its partial peptide,or a salt thereof,

(2) the agent according to the above (1), wherein the Otx2 protein is aprotein comprising the same amino acid sequence as, or substantially thesame amino acid sequence as an amino acid sequence represented by SEQ IDNO: 1, SEQ ID NO: 3 or SEQ ID NO: 5,(3)

(3) an agent for preventing, treating or suppressing progression of aretinal disease, which comprises a DNA or an RNA encoding an Otx2protein or its partial peptide,

(4) the agent according to the above (3), wherein the DNA comprises anucleotide sequence represented by SEQ ID NO: 2 , SEQ ID NO: 4 or SEQ IDNO: 6, or a nucleotide sequence which hybridizes with the said sequenceunder highly stringent conditions,

(5) the agent according to the above (3), which comprises a recombinantvector containing a DNA or an RNA encoding an Otx2 protein or itspartial peptide,

(6) an agent for inducing differentiation into a retinal photoreceptorcell, which comprises an Otx2 protein or its partial peptide, or a saltthereof,

(7) an agent for inducing differentiation into a retinal photoreceptorcell, which comprises a DNA or an RNA encoding an Otx2 protein or itspartial peptide,

(8) a method for inducing differentiation into a retinal photoreceptorcell, which comprises expressing an Otx2 protein, or increasing anamount of expression of an Otx2 protein,

(9) the method for inducing differentiation into a retinal photoreceptorcell according to the above (8), which comprises expressing an Otx2protein, or increasing an amount of expression of an Otx2protein in aneye ball tissue-derived cell, an embryonic stem cell, a neural stem cellor a neural precursor cell,

(10) the method for inducing differentiation into a retinalphotoreceptor cell according to the above (8), wherein a DNA or an RNAencoding an Otx2 protein or its partial peptide is introduced into aneye ball tissue-derived cell, an embryonic stem cell, a neural stem cellor a neural precursor cell, and the resulting cell is cultured,

(11) a method for preventing, treating or suppressing progression of aretinal disease, which comprises administering an effective amount ofOtx2 protein or its partial peptide, or a salt thereof to a mammal,

(12) a method for preventing, treating or suppressing progression of aretinal disease, which comprises administering an effective amount of aDNA or an RNA encoding an Otx2 protein or its partial peptide to amammal,

(13) a method for regenerating retina, which comprises transplanting aretinal photoreceptor cell or a precursor cell thereof which isdifferentiation-induced by an Otx2 protein, into retina,

(14) use of an Otx2 protein or its partial peptide, or a salt thereoffor preparing an agent for preventing, treating or suppressingprogression of a retinal disease,

(15) use of a DNA or an RNA encoding an Otx2 protein or its partialpeptide for preparing an agent for preventing, treating or suppressingprogression of a retinal disease.

(16) a diagnostic agent for a retinal disease, which comprises anantibody to an Otx2 protein or its partial peptide, or a salt thereof,

(17) a method for diagnosing a retinal disease, which comprises using anantibody to an Otx2 protein or its partial peptide, or a salt thereof,

(18) a method for diagnosing a retinal disease, which comprisesdetecting an expression amount or a mutation of an Otx2 protein or itspartial peptide,

(19) a diagnostic agent for a retinal disease, which comprises (a) a DNAor an RNA encoding an Otx2 protein or a partial peptide, or (b) anantisense polynucleotide comprising a nucleotide sequence complementaryor substantially complementary to a nucleotide sequence of the said DNAor RNA,

(20) a method for diagnosing a retinal disease, which comprises using(a) a DNA or an RNA encoding an Otx2 protein or its partial peptide, or(b) an antisense polynucleotide comprising a nucleotide sequencecomplementary or substantially complementary to a nucleotide sequence ofthe said DNA or RNA,

(21) a method for screening a compound having an action of inducingdifferentiation into a retinal photoreceptor cell, or a salt thereof,which comprises using, as an index, an expression of, or increase in anexpression amount of an Otx2 protein or its partial peptide,

(22) the screening method according to the above (21), wherein a cellhaving an ability of expressing an Otx2 protein or its partial peptideis used,

(23) a kit for screening a compound having an action of inducingdifferentiation into a retinal photoreceptor cell, or a salt thereof,which comprises a cell having an ability of expressing an Otx2 proteinor its partial peptide,

(24) a compound having an action of inducing differentiation into aretinal photoreceptor cell, or a salt thereof, which is obtainable usinga screening method as defined in the above (21) or (22), or a screeningkit as defined in the above (23), and

(25) a medicine, which comprises a compound as defined in the above (24)or a salt thereof.

Using the protein of the present invention or the DNA of the presentinvention, an eye ball tissue-derived cell, an embryonic stem cell, aneural stem cell or a neural precursor cell can be differentiated into aretinal photoreceptor cell. Therefore, by regenerating or newlyproducing a retinal photoreceptor cell using the protein of the presentinvention or the DNA of the present invention, retinal diseases such asretinitis pigmentosa, senile macular degeneration, diabetic retinopathy,retinal detachment, glaucoma and retinal vessel occlusion can beprevented or treated, or progression of such disease can be suppressed.Further, since retinal photoreceptor cells undergo structural orfunctional abnormalities due to abnormality of an Otx2 gene, diagnosisof such retinal diseases can be performed by detecting abnormality of anOtx2 gene, or degeneration or reduction in expression of an Otx2protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing the structure of a control virus vector and anOtx2 virus vector. In the figure, AP represents a placenta-derivedalkaline phosphatase gene, and LTR represents a virus promoter. WhenOtx2 virus vector has IRES sequence between Otx2 and AP, the vectorallows for coexpression of both genes (Otx2 and AP). The IRES sequenceis a virus-derived gene.

FIG. 2 is a view showing summary of a virus infection test. In thefigure, “Retina” represents retina, “Pigment Epithelium” representsretinal pigment epithelium, “virus” represents a range infected with avirus, “Remove Retina” represents extraction of retina, “Fix and Stain”represents tissue fixation and staining, and “Section and AnalyseClones” represents a frozen section and its analysis.

FIG. 3 is a view showing an example of a retinal frozen section imageafter alkaline phosphatase staining. In the figure, “Bi” represents abipolar cell, “A” represents an amacrine cell, “R” represents a retinalphotoreceptor cell, “M” represents a Muller glial cell, “R+Bi”represents a retinal photoreceptor cell and a Muller glial cell, and“A+Bi” represents an amacrine cell and a bipolar cell.

FIG. 4 is a view showing an existence ratio of each of cellsdifferentiated from a retinal stem cell infected with a control virusvector (LIA) or an Otx2 virus vector (Otx2/LIA), relative to a totalcell number in a microscopic one field.

BEST MODE FOR CARRYING OUT THE INVENTION

Examples of Otx2 proteins used in the present invention include aprotein containing the same or substantially the same amino acidsequence as an amino acid sequence represented by SEQ ID NO: 1, 3 or 5.In the present invention, the Otx2 proteins are not limited to ahuman-derived Otx2 protein having an amino acid sequence represented bySEQ ID NO: 1 or 3, and a mouse-derived Otx2 protein having an amino acidsequence represented by SEQ ID NO: 5, but may have an amino acidsequence derived from other animals, particularly, a warm-blooded animal(e.g. guinea pig, mouse, chicken, rabbit, pig, sheep, cow, monkey,etc.), or substantially the same amino acid sequence as the saidsequence.

Examples of the “amino acid sequence substantially the same as an aminoacid sequence represented by SEQ ID NO: 1, 3 or 5” include amino acidsequences having not less than about 50%, preferably not less than about60%, more preferably not less than about 70%, further preferably notless than about 80%, inter alia, preferably not less than about 90%,most preferably not less than about 95% homology with an amino acidsequence represented by SEQ ID NO: 1, 3 or 5. As a protein containing anamino acid sequence which is substantially the same as an amino acidsequence represented by SEQ ID NO: 1, 3 or 5 of the present invention,for example, a protein containing an amino acid sequence which issubstantially the same as an amino acid sequence represented by SEQ IDNO: 1, 3 or 5, and having an action of inducing differentiation into aretinal photoreceptor cell is preferable. Inter alia, it is preferablethat transcription activity, and action of inducing differentiation intoa retinal photoreceptor cell are equivalent to a protein having an aminoacid sequence represented by SEQ ID NO: 1, 3 or 5 (e.g. about 0.01 to100 fold, preferably about 0.5 to 20 fold, more preferably about 0.5 to2 fold), and extent of such activity or quantitative elements such asmolecular weight of proteins may differ. Measurement of action ofinducing differentiation into a retinal photoreceptor cell can beperformed according to the known method, for example, such action can bemeasured according to a screening method described later. Measurement oftranscription activity can be performed using the known method such asreporter assay and RT-PCR.

Specifically, as an Otx2 protein used in the present invention, there isused a protein containing (a) an amino acid sequence in which one or twoor more (preferably, around 1 to 30, more preferably around 1 to 40,still more preferably several (1 to 5)) of amino acids in an amino acidsequence represented by SEQ ID NO: 1, 3 or 5 are deleted, (b) an aminoacid sequence in which one or two or more (preferably, around 1 to 30,more preferably around 1 to 10, still more preferably several (1 to 5))of amino acids are added to an amino acid sequence represented by SEQ IDNO: 1, 3 or 5, (c) an amino acid sequence in which one or two or more(preferably, around 1 to 30, more preferably around 1 to 10, still morepreferably several (1 to 5)) of amino acids in an amino acid sequencerepresented by SEQ ID NO: 1, 3 or 5 are substituted with other aminoacids, or (d) an amino acid sequence as a combination thereof. The aminoacid which is added in the (b), and the amino acid which is substitutedin the (c) may be a non-natural amino acid other than 20 kinds of aminoacids encoded by a gene. It is more preferable that the proteindescribed in the (a) to (d) has an action of inducing differentiationinto a retinal photoreceptor cell.

In the Otx2 proteins of the present invention, the C-terminus may be anyone of carboxyl group (—COOH), carboxylate (—COO⁻), amide (—CONH₂) andester (—COOR). Herein, as R in the ester group, there are used a C₁₋₆alkyl group such as methyl, ethyl, n-propyl, isopropyl and n-butyl; aC₃₋₈ cycloalkyl group such as cyclopentyl and cyclohexyl; a C₆₋₁₂ arylgroup such as phenyl and α-naphthyl; and a C₇₋₁₄ aralkyl group such as aphenyl-C₁₋₂ alkyl group (e.g. benzyl and phenethyl) and anα-naphthyl-C₁₋₂ alkyl group (e.g. α-naphthylmethyl); and additionally, apivaloyloxymethyl group which is generally used as an oral ester. Whenthe Otx2 protein in the present invention has a carboxyl group (orcarboxylate) at a position other than the C-terminus, the protein inwhich a carboxyl group is amidated or esterified is also included in theOtx2 protein of the present invention. In this case, as the ester, forexample, the aforementioned C-terminal ester is used. Further, the Otx2protein in the present invention includes a protein in which the aminogroup of N-terminal methionine residue is protected with a protectinggroup (e.g. C₁₋₆ acyl group such as C₂₋₆ alkanoyl group including, forexample, formyl and acetyl), a protein in which a glutamyl groupproduced by cleavage at the N-terminal in a living body is convertedinto pyroglutamic acid, a protein in which a substitutable group (e.g.—OH, —SH, amino group, imidazole group, indole group, guanidine group,etc.) on a side chain of an amino acid in the molecule is protected withan appropriate protecting group (e.g. C₁₋₆ acyl group such as C₂₋₆alkanoyl group including, for example, formyl group and acetyl), and aso-called conjugated protein such as glycoprotein to which a sugar chainis bound.

As a partial peptide of Otx2 proteins used in the present invention(hereinafter, abbreviated as partial peptide in some cases), any one maybe used as far as it is a partial peptide of the Otx2 protein. As thenumber of amino acids of such partial peptide in the present invention,a peptide containing an amino acid sequence of at least about not lessthan 20, preferably about not less than 50, or more preferably about notless than 100 amino acids among a constitutional amino acid sequence ofthe Otx2 protein is preferable.

In a partial peptide of the present invention, the C-terminus may be anyone of carboxyl group (—COOH), carboxylate (—COO⁻), amide (—CONH₂) andester (—COOR). Further, the partial peptide of the present inventionincludes, like the Otx2 protein of the present invention, a partialpeptide wherein the amino group of methionine residue at the N-terminusis protected with a protecting group, a partial peptide wherein Glnproduced by cleavage of the N-terminal side in a living body isconverted into pyroglutamic acid, a partial peptide wherein asubstituent on the side chain of an amino acid in the molecule isprotected with an appropriate protecting group, and a conjugated peptidesuch as a so-called glycopeptide to which a sugar chain is bound.

Examples of a salt of the Otx2 protein of the present invention or apartial peptide thereof include physiologically acceptable salts with anacid or a base. Inter alia, physiologically acceptable acid additionsalts are preferable. Examples of such salts include salts withinorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromicacid, sulfuric acid), or with organic acids (e.g. acetic acid, formicacid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaricacid, citric acid, malic acid, oxalic acid, benzoic acid, methansulfonicacid, benzene sulfonic acid).

The Otx2 protein in the present invention or a salt thereof can beprepared from a cell or a tissue of an animal, preferably a warm-bloodedanimal, more preferably human or rat, further preferably human,particularly preferably human brain neuron, ocular retinal pigmentepithelial cell or a retinal photoreceptor cell by the known method ofpurifying a protein, or can also be prepared by culturing a transformantcontaining a DNA or an RNA encoding the Otx2 protein of the presentinvention described later. Alternatively, such protein can also beprepared in the same or similar manner to a protein synthesis methoddescribed later. When the protein is prepared from animal tissues orcells, animal tissues or cells are homogenized, extracted with an acid,and the extract can be purified and isolated by combiningchromatographies such as reverse phase chromatography, and ion exchangechromatography.

For synthesizing an Otx2 protein in the present invention or a partialpeptide thereof or a salt thereof or an amide thereof, a commerciallyavailable protein synthesis resin can be usually used. Examples of suchresin include a chloromethyl resin, a hydroxymethyl resin, abenzhydrylamine resin, an aminomethyl resin, a 4-benzyloxybenzyl alcoholresin, a 4-methylbenzhydrylamine resin, a PAM resin, a 4-hydroxymethylmethylphenylacetamido methyl resin, a polyacrylamide resin, a4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin, and a4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl)phenoxy resin. Using suchresin, amino acids in which an α-amino group, and a side chainfunctional group are appropriately protected are condensed in the orderof the sequence of desired proteins on a resin by the known variouscondensing methods. At the end of the reaction, the protein is cleavedfrom the resin, and at the same time, various protecting groups areremoved, and further, an intramolecular disulfide bond forming reactionis performed in a highly diluted solution, thereby to obtain a desiredprotein or an amide thereof. Regarding the aforementioned condensationof protected amino acids, various activating reagents which can be usedin synthesizing proteins can be used. As an activating reagent,carbodiimides are particularly preferable. As the carbodiimides used,there are exemplified DCC, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminoprolyl)carbodiimide. In the activation bythese reagents, the protected amino acid may be added to a resin afteractivation of a protected amino acid is performed in advance by adding aprotected amino acid directly to a resin together with a racemizationsuppressing additive (e.g. HOBt, HOOBt), or by converting a protectedamino acid into symmetric acid anhydride or HOBt ester or HOOBt ester.

A solvent used in activation of a protected amino acid or condensationwith a resin can be appropriately selected from solvents which are knownto be usable in a protein condensing reaction. Such solvents include,for example, acid amides such as N,N-dimethylformamide,N,N-dimethylacetamide, and N-methylpyrrolidone; halogenated hydrocarbonssuch as methylene chloride, and chloroform; alcohols such astrifluoroethanol; sulfoxides such as dimethyl sulfoxide; ethers such aspyridine, dioxane and tetrahydrofuran; nitriles such as acetonitrile,and propionitrile; esters such as methyl acetate and ethyl acetate, andan appropriate mixture thereof. The reaction temperature isappropriately selected from a range which is known to be usable in aprotein bond formation, and is usually appropriately selected from arange of −20° C. to 50° C. An activated amino acid derivative is usuallyused in 1.5 to 4-fold excessive amount. When condensation is found to beinsufficient as a result of a test using a ninhydrine reaction, it ispossible to perform sufficient condensation by repeating a condensingreaction without removal of protecting groups. When sufficientcondensation is not resulted even after repeated reactions, unreactedamino acids may be acetylated using acetic anhydride or acetylimidazole.

Examples of protecting groups used for an amino group of a raw materialinclude, for example, Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl,trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl,diphenylphosphinothioyl, and Fmoc. A carboxyl group can be protected,for example, by alkyl esterification (e.g. linear, branched, or cyclicalkyl esterification such as methyl, ethyl, propyl, butyl, t-butyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and 2-adamantylesters), aralkyl esterification (e.g. benzyl ester, 4-nitrobenzyl ester,4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl ester), phenacylesterification, benzyloxycarbonyl hydrazide formation, t-butoxycarbonylhydrazide formation, or trityl hydrazide formation. The hydroxy group ofserine can be protected, for example, by esterification oretherification. As a group suitable for this esterification, forexample, a lower alkanoyl group such as an acetyl group, an aroyl groupsuch as a benzoyl group, and a group derived from carbonic acid such asa benzyloxycarbonyl group and an ethoxycarbonyl group are used. Inaddition, a group suitable for etherification is, for example, a benzylgroup, a tetrahydropyranyl group, or a t-butyl group. As a protectinggroup for the phenolic hydroxy group of tyrosine, for example, Bzl,Cl₂-Bzl, 2-nitrobenzyl, Br-Z, and t-butyl are used. As a protectinggroup for the imidazole of histidine, for example, Tos,4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum,Boc, Trt and Fmoc are used.

As an activated carboxyl group of a raw material, for example,corresponding acid anhydride, azide, and active ester [ester withalcohol (e.g. pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt)] are used. As anactivated amino group of a raw material, for example, correspondingphosphoric acid amide is used. As a method of removing (deprotecting) aprotecting group, there are used, for example, (a) catalytic reductionin a hydrogen stream in the presence of a catalyst such as Pd-black orPd-carbon, (b) acid treatment with anhydrous hydrogen fluoride,methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroaceticacid or a mixed solution thereof, (c) base treatment withdiisopropylethylamine, triethylamine, piperidine, or piperazine, or (d)reduction with sodium in liquid ammonia. The deprotection by theabove-mentioned acid treatment is generally performed at a temperatureof about −20° C. to 40° C., but in such acid treatment, it is effectiveto add a scavenger for cations, such as anisole, phenol, thioanisole,m-cresol, p-cresol, dimethyl sulfide, 1,4-butanedithiol, and1,2-ethanedithiol. The 2,4-Dinitrophenyl group used as a protectinggroup for the imidazole of histidine is removed by thiophenol treatment.The formyl group used as a protecting group for the indole of tryptophanmay also be removed by alkali treatment with dilute sodium hydroxidesolution, dilute ammonia or the like, in addition to deprotection byacid treatment in the presence of 1,2-ethanedithiol or1,4-butanedithiol.

Protection of functional groups which should not be involved in thereaction of raw materials, and protecting groups therefor, anddeprotection of such protecting groups, and activation of the functionalgroups involved in the reaction can be appropriately selected from theknown groups or the known means. As an alternative method of obtainingprotein amides, for example, an α-carboxyl group of carboxyl terminalamino acid is first protected by amidation, a peptide (protein) chain isextended at the amino group side to a desired chain length, a protein inwhich only a protecting group for an α-amino group at the N-terminus ofthe peptide chain is removed, and a protein in which only a protectinggroup for the carboxyl group at the C-terminus is removed are prepared,and both these proteins are condensed in the aforementioned mixedsolvent. Details of the condensation reaction are the same as describedabove. After the protected protein obtained by condensation is purified,all protecting groups can be removed by the aforementioned method toobtain desired crude protein. This crude protein is purified by usingvarious known purification means, and main fractions are lyophilized,thereby to give a desired protein amide. For obtaining an ester entityof proteins, an α-carboxyl group of carboxyl terminal amino acids iscondensed with a desired alcohol to obtain an amino acid ester, and thena desired ester of proteins can be obtained similarly according to theprocedure in the preparation of protein amides. When a protein obtainedby the aforementioned method is a free compound, it can be convertedinto an appropriate salt by the known method, and conversely, when theprotein is obtained as a salt, it can be converted into a free compoundby the known method.

A partial peptide or a salt of the Otx2 protein in the present inventioncan be prepared according to the known peptide synthesis method, or bycleavage of the Otx2 protein in the present invention with anappropriate peptidase. As a method of synthesizing a peptide, forexample, any one of a solid phase synthesis method and a liquid phasesynthesis method may be used. That is, a desired peptide can be preparedby condensing a partial peptide or an amino acid which can constitutethe Otx2 protein of the present invention, with a remaining part, andremoving the protecting group when the product has a protecting group.Examples of the known condensing method and deprotection of protectinggroups include methods described, for example, in the following (a) to(e): (a) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, IntersciencePublishers, New York (1966), (b) Schroeder and Luebke, The Peptide,Academic Press, New York (1965), (c) Nobuo Izumiya et al., Base andexperiment of peptide synthesis, Maruzen (1975), (d) Haruaki Yajima andShunpei Sakakibara, Biochemistry Experimental Course 1, Chemistry ofProtein IV, 205, (1977), and (e) Development of Medicines, vol. 14,Sequel, Peptide Synthesis, supervised by Haruaki Yajima, Hirokawashoten.

In addition, after the reaction, the partial peptide of the presentinvention can be purified and isolated by a conventional purificationmethod, for example, in combination of solventextraction/distillation/column chromatography/liquidchromatography/recrystallization. When the partial peptide obtained bythe aforementioned method is a free compound, it can be converted intoan appropriate salt by the known method, and conversely, when it isobtained as a salt, it can be converted into a free compound by theknown method.

As a DNA encoding an Otx2 protein used in the present invention, any oneof a genomic DNA, a genome DNA library, a cDNA derived from theaforementioned cells/tissues, a cDNA library derived from theaforementioned cells/tissues, and a synthetic DNA may be used. A vectorused in a library may be any one of bacteriophage, plasmid, cosmid, andphagemide. In addition, a DNA can also be amplified directly by ReverseTranscriptase Polymerase Chain Reaction (hereinafter, abbreviated asRT-PCR method) using a total RNA or mRNA fraction prepared from theaforementioned cells/tissues. Specifically, examples of a DNA encodingan Otx2 protein include (a) a DNA comprising a nucleotide sequencerepresented by SEQ ID NO: 2, 4 or 6, and (b) a DNA having a nucleotidesequence which hybridizes with a nucleotide sequence represented by SEQID NO: 2, 4 or 6 under highly stringent conditions, and encoding an Otx2protein having substantially the same quality of activity (e.g.transcription activity, retinal photoreceptor cell differentiationinducing action, etc.) as that of the Otx2 protein of the presentinvention. SEQ ID NOS: 2 and 4 are a DNA encoding a human Otx2 protein(Kastury, K., et al., “Chromosome locations of human EMX and OTX genes”,Genomics 22 (1), 41-45 (1994)), and SEQ ID NO: 6 is a DNA encoding amouse Otx2 protein (Simeone, A., et al., “Nested expression domains offour homeobox genes in developing rostral brain”, Nature 358 (6388),687-690 (1992)). As a DNA which can hybridize with a nucleotide sequencerepresented by SEQ ID NO: 2, 4 or 6, for example, there is used a DNAcomprising a nucleotide sequence having about not lower than 70%homology, preferably about not lower than 80% homology, more preferablyabout not lower than 90% homology, most preferably about not less than95% homology, with a nucleotide sequence represented by SEQ ID NO: 2, 4or 6. Hybridization can be performed according to the known method or asimilar method thereof, for example, the method described in Molecularcloning 2^(nd) J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989).In addition, when a commercially available library is used,hybridization can be performed according to the method described in theinstruction manual attached thereto. More preferably, such hybridizationcan be performed according to highly stringent conditions. The highlystringent conditions indicate, for example, conditions of a sodiumconcentration of about 19 to 40 mM, preferably about 19 to 20 mM, and atemperature of about 50 to 70° C., preferably about 60 to 65° C. Inparticular, most preferable conditions are that the sodium concentrationis about 19 mM and the temperature is about 65° C.

As a DNA encoding a partial peptide used in the present invention, anyDNA comprising a nucleotide sequence encoding the aforementioned partialpeptide of the present invention may be used. Alternatively, any one ofa genomic DNA, a genome DNA library, a cDNA derived from theaforementioned cells/tissues, a cDNA library derived from theaforementioned cells/tissues, and a synthetic DNA may be used. A vectorused in a library may be any one of bacteriophage, plasmid, cosmid, andphageimide. In addition, a DNA may be amplified directly by ReverseTranscriptase Polymerase Chain Reaction (hereinafter, abbreviated asRT-PCR method) using an mRNA fraction prepared from the aforementionedcells/tissues. Specifically, as a DNA encoding a partial peptide of thepresent invention, for example, such DNA to be used includes, forexample, (a) a DNA containing a partial nucleotide sequence of a DNAcomprising a nucleotide sequence represented by SEQ ID NO: 2, 4 or 6,(b) a DNA containing a nucleotide sequence which hybridizes with a DNAcomprising a nucleotide sequence represented by SEQ ID NO: 2, 4 or 6under highly stringent conditions, and encoding a protein havingsubstantially the same quality of activity (e.g. transcription activity,retinal photoreceptor cell differentiation inducing action, etc. ) asthat of the Otx2 protein of the present invention, and a DNA containinga partial nucleotide sequence of the (a) or (b).

Although an RNA encoding an Otx2 protein or its partial peptide used inthe present invention is not particularly limited as far as it canexpress an Otx2 protein or its partial peptide by a transcriptase, itcan be obtained by the known means.

As a means for cloning a DNA completely encoding an Otx2 protein in thepresent invention or a partial peptide thereof (hereinafter, abbreviatedas present protein in some cases), the DNA can be amplified by using asynthetic DNA primer containing a partial nucleotide sequence of aprotein of the present invention by a PCR method, or can be selectedfrom a DNA incorporated into an appropriate vector, by hybridizationusing a DNA fragment or a synthetic DNA encoding a part or an entireregion of the labeled present protein. A method of hybridization can beperformed according to the method described, for example, in MolecularCloning 2^(nd) J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989.In addition, when a commercially available library is used,hybridization can be performed according to the method described in theinstruction manual attached thereto.

Substitution of a nucleotide sequence of a DNA can be performed usingPCR or the known kit, for example, Mutan™-superExpress Km (TAKARA SHUZOCO., LTD.), or Mutan™-K (TAKARA SHUZO CO., LTD.) by the known methodsuch as the ODA-LAPCR method, the gapped duplex method, and the Kunkelmethod, or a similar method thereof. The cloned DNA encoding a proteinof the present invention can be used as it is, depending on the purpose,or can be used, if desired, by digesting with a restriction enzyme or byadding a linker. The DNA has ATG as a translation initiation codon atthe 5′-end, and has TAA, TGA or TAG as a translation termination codonat the 3′-end. These translation initiation codon and translationtermination codon may be added using an appropriate synthetic DNAadaptor.

A DNA or an RNA encoding the protein of the present invention(hereinafter, abbreviated as present DNA, in some cases) may be modifiedbased on the following strategy, i.e. to more stabilize the present DNAin a cell, to enhance the cell permeability of the present DNA, and toreduce the toxicity of the present DNA, if any. Many modifications likethese are known in the art, and are disclosed, for example, in J.Kawakami et al, Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8. pp.395, 1992; S. T. Crooke et al. ed., Antisense Research and Applications,CRC Press, 1993. The present DNA may be used in a special form in whichthe DNA is encapsulated in a liposome or a microsphere. In addition,other substances other than a base may be added to the present DNA.Examples of other substances include a sugar; an acid or a base; apolycation compound such as polylysine acting to neutralize a charge ofa phosphate nucleus; and a hydrophobic substance such as lipid (e.g.phospholipid, cholesterol, etc.) which enables to enhance interactionwith a cell membrane, or to increase uptake of nucleic acids. Examplesof preferable lipids to be added include cholesterol and a derivativethereof (e.g. cholesteryl chloroformate, cholic acid, etc.). Othersubstance described above can be attached to the 3′-end or 5′-end ofnucleic acids, and can be attached via a base, a sugar, or anintramolecular nucleoside bond. The present DNA may be chemicallymodified at the terminus. Examples of groups for such terminalmodification include a group for cap specifically arranged at 3′-end or5′-end of nucleic acids, which suppress degradation due to nuclease suchas exonuclease, and RNase. Examples of such groups for cap include ahydroxy-protecting group known in the art, including glycols such aspolyethylene glycol and tetraethylene glycol, though they are notlimited thereto.

As a recombinant vector containing a DNA or RNA encoding the Otx2protein or its partial peptide used in the present invention, anexpression vector capable of expressing an Otx2 protein or its partialpeptide is preferable.

An expression vector of the protein of the present invention can beprepared by (i) excising a DNA fragment containing a DNA encoding thepresent protein from, for example, cDNA and (ii) ligating said DNAfragment in the downstream direction of a promoter in an appropriateexpression vector.

As the above expression vector, there are used Escherichia coli-derivedplasmid (e.g. pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13), Bacillussubtilis-derived plasmid (e.g. pUB110, pTP5, pC194), yeast-derivedplasmid (e.g. pSH19, pSH15), bacteriophage such as λ phage, virus suchas retrovirus, adenovirus, lentivirus, vaccinia virus, and baculovirusand, additionally, pA1-11, pXT1, pRc/CMV, pRc/RSV, and pcDNAI/Neo. Interalia, as a vector used in the present invention, a virus is preferable,and retrovirus, adenovirus and lentivirus are more preferable.

As the promoter described above, any promoter may be used as far as itis an appropriate promoter corresponding to a host used in theexpression of the gene. For example, when an animal cell is used as ahost, examples of such promoters include SRα promoter, SV40 promoter LTRpromoter, CMV promoter, and HSV-TK promoter. Among them, it ispreferable to use LTR promoter, CMV promoter, or SRα promoter. When ahost is a bacterium of the genus Escherichia, trp promoter, lacpromoter, recA promoter, λP_(L) promoter, and lpp promoter arepreferable. When a host is a bacterium of the genus Bacillus, SPO1promoter, SPO2 promoter, and penP promoter are preferable. When a hostis yeast, PHO5 promoter, PGK promoter, GAP promoter, and ADH promoterare preferable. When a host is an insect cell, polyhedrin promoter, andP10 promoter are preferable.

As an expression vector, expression vectors containing optionally, inaddition to the above elements, an enhancer, a splicing signal, a poly Aaddition signal, a cap structure, a protein synthesis initiation signal,a selectable marker, a labeled marker, and SV40 replication origin canbe used.

Examples of the selectable marker include dihydrofolate reductase(hereinafter, abbreviated as dhfr in some cases) gene [methotrexate(MTX) resistance], ampicillin resistant gene (hereinafter, abbreviatedas Amp^(r) in some cases), and neomycin resistant gene (hereinafter,abbreviated as Neo^(r) in some cases, G418 resistance). In particular,when a dhfr gene is employed as a selectable marker using dhfrgene-deficient Chinese hamster cell CHO, an objective gene may beselected on a medium containing no thymidine.

As the labeled marker, an alkaline phosphatase (hereinafter, abbreviatedas AP in some cases) gene, and green fluorescent protein (GFP) gene canbe preferably used.

In addition, a signal sequence suitable for a host may be optionallyadded to an expression vector. When a host is a bacterium of the genusEscherichia, PhoA signal sequence, and OmpA signal sequence can beutilized. When a host is a bacterium of the genus Bacillus, α-amylasesignal sequence, and subtilisin signal sequence can be utilized. When ahost is yeast, MFα signal sequence, and SUC2 signal sequence can beutilized. When a host is an animal cell, insulin signal sequence,α-interferon signal sequence, and antibody molecule signal sequence canbe utilized.

When a virus is used as a vector, in order to enhance translationmechanism, it is preferable to arrange an IRES (interiorribosome-binding site) sequence as a protein synthesis initiationsignal.

By introducing into a host the thus constructed expression vectorcontaining a DNA encoding the protein of the present invention, atransformant can be prepared.

As a host, for example, Escherichia bacteria, Bacillus bacteria, yeasts,insect cells, insects, and animal cells are used. As an embodiment ofEscherichia bacteria, there are used Escherichia coli K12/DH1 (Proc.Natl. Aced. Sci. USA, vol. 60, 160 (1968)), JM103 (Nucleic acidsResearch, vol. 9, 309 (1981)), JA221 (Journal of Molecular Biology vol.120, 517 (1978)), HB101 (Journal of Molecular Biology, vol. 41, 459(1969)), C600 (Genetics, vol. 39, 440 (1954)), DH5α (Inoue, H., Nojima,H. and Okayama, H., Gene, 96, 23-28 (1990)), and DH10B (Proc. Natl.Acad. Sci. USA, vol. 87, 4645-4649 (1990)). As Bacillus bacteria, forexample, Bacillus subtilis MI114 (Gene, vol. 24, 255 (1983)), and 207-21(Journal of Biochemistry, vol. 95, 87 (1984)) are used. As yeast, forexample, Saccharomyces cerevisiae AH22, AH22R⁻, NA87-11A, DKD-5D,20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, and Pichiapastoris.

As an insect cell, for example, when a virus is AcNPV, there are usedcabbage armyworm larva-derived established cell (Spodoptera frugiperdacell; Sf cell), MG1 cell derived from Trichoplusia ni midgut, High Five™cell derived from Trichoplusia ni egg, Mamestrabrassicae-derived celland Estigmena acrea-derived cell. When a virus is BmNPV,silkworm-derived established cell (Bombyx mori N; BmN cell) is used. Asthe said Sf cell, for example, Sf9 cell (ATCC CRL1711), and Sf21 cell(Vaughn, J. L. et al, In Vivo, 13, 213-217, (1977)) are used. As aninsect, for example, silkworm larva is used (Maeda et al., Nature, vol.315, 592 (1985)). As an animal cell, for example, monkey cell (COS-7,Vero, Chinese Hamster cell CHO (hereinafter, abbreviated as CHO cell),dhfr gene-deficient Chinese Hamster cell CHO (hereinafter, abbreviatedas CHO (dhfr) cell), mouse L cell, mouse AtT-20, mouse myeloma cell, ratGH3 , and human FL cell are used.

In order to transform Escherichia bacteria, transformation can bepreformed according to the method described, for example, in Proc. Natl.Acad. Sci. USA, vol. 69, 2110 (1972) or Gene, vol. 17, 107 (1982). Inorder to transform Bacillus bacteria, transformation can be performedaccording to the method described, for example, in Molecular & GeneralGenetics, vol. 168, 111 (1979). In order to transform yeast,transformation can be preformed according to the method described, forexample, in Methods in Enzymology, vol. 194, 182-187 (1991), Proc. Natl.Acad. Sci. USA, vol. 75, 1929 (1978). In order to transform insect cellsor insects, transformation can be performed according to the methoddescribed, for example, in Bio/Technology, vol. 6, 47-55 (1988). Inorder to transform animal cells, transformation can be preformedaccording to the method described, for example, in Cell Technologyseparate volume 8 New Cell Technology Experimental Protocol, 263-267(1995) (published by Shujunsha), Virology, vol. 52, 456 (1973). Likethis, a transformant transformed with an expression vector containing aDNA encoding a protein of the present invention is obtained. When atransformant of which host is Escherichia bacterium or Bacillusbacterium is cultured, a liquid medium is suitable for a culture medium,and a carbon source, a nitrogen source, an inorganic substance and otheradditives which are required to grow a transformant are containedtherein. Examples of the carbon source include glucose, dextrin, solublestarch, and sucrose, examples of the nitrogen source include inorganicor organic substances such as ammonium salts, nitrate salts, corn steepliquor, peptone, casein, meat extract, soybean meal, and potato extract,and examples of inorganic substances include calcium chloride, sodiumdihydrogen phosphate, and magnesium chloride. In addition, yeastextract, vitamins, and growth promoting factor may be added. Medium pHis desirably about 5 to 8.

As a medium upon culturing of Escherichia bacteria, for example, M9medium containing glucose and casamino acid (Miller, Journal ofExperiments in Molecular Genetics, 431-433, Cold Spring HarborLaboratory, New York, 1972) is preferable. Herein, in order to make apromoter act effectively, for example, a drug such as 3β-indolylacrylicacid may be added, if required. When a host is Escherichia bacterium,culturing is performed usually at about 15° C. to 43° C. for about 3 to24 hours, and if necessary, aeration or stirring may be carried out.When a host is Bacillus bacterium, culturing is usually performed atabout 30 to 40° C. for 6 to 24 hours, and if necessary, aeration orstirring may be performed. When a transformant of which host is yeast iscultured, examples of a medium include Burkholder minimum medium(Bostian, K. L. et al, Proc. Natl. Acad. Sci. USA, vol. 77, 4505(1980)), and a SD medium containing 0.5% casamino acid (Bitter, G. A. etal., Proc. Natl. Acad. Sci. USA, vol. 81, 5330 (1984)). It is preferableto adjust the pH of the medium to about 5 to 8. Culturing is usuallyperformed at 20° C. to 35° C. for about 24 to 72 hours, and ifnecessary, aeration or stirring may be optionally performed.

When a transformant of which host is an insect cell or an insect iscultured, there is used, as a medium, a medium in which an additive suchas immobilized 10% bovine serum is appropriately added to Grace's InsectMedium (Grace T. C. C., Nature, vol. 195, 788 (1962)). It is preferableto adjust the pH of the medium to about 6.2 to 6.4. Culturing isperformed usually at about 27° C. for about 3 to 5 days, and ifnecessary, aeration or stirring may be optionally performed. When atransformant of which host is an animal cell is cultured, there are usedas a medium, for example, MEM medium containing about 5 to 20% fetalbovine serum (Science, vol. 122, 501 (1952)), DMEM medium (Virology,vol. 8, 396 (1959)), RPMI 1640 medium (The Journal of the AmericanMedical Association, vol. 199, 519 (1967)), and 199 medium (Proceedingof the Society for the Biological Medicine, vol. 73, 1 (1950)) are used.It is preferable that the pH is about 6 to 8. Culturing is usuallyperformed at 30° C. to 40° C. for about 15 to 60 hours, and ifnecessary, aeration or stirring may be performed. As described above,the protein of the present invention can be produced in a cell, in acell membrane or outside a cell of a transformant.

In order to separate and purify the protein of the preset invention fromthe culture broth, for example, separation and purification can beperformed by the following method. When the protein of the presentinvention is extracted from cultured bacterial cells or cells, there areappropriately employed a method of colleting bacterial cells or cells bythe known method after culturing, suspending these cells in anappropriate buffer, disrupting the suspended cells by ultrasound,lysozyme and/or freezing/melting, and obtaining the crude extract of theprotein by centrifugation or filtration, and the like. A proteindegenerating agent such as urea and guanidine hydrochloride, or asurfactant such as Triton X-100™ may be contained in a buffer. When aprotein is secreted in a culture solution, after completion of theculturing, bacterial cells or cells and the culture supernatant areseparated by the known method, and the culture supernatant is collected.Purification of a protein contained in the resulting extract or theculture supernatant can be performed by appropriate combination with theknown separation/purification methods. As the known separation orpurification method, there are used a method of utilizing solubilitysuch as salting out and solvent precipitation method; a method ofutilizing mainly differences in a molecular weight of a dialysis method,an ultrafiltration method, a gel filtration method, and anSDS-polyacrylamide gel electrophoresis method; a method of utilizingdifferences in charge such as ion exchange chromatography; a method ofutilizing specific affinity such as affinity chromatography; a method ofutilizing differences in hydrophobicity such as reverse phase highperformance liquid chromatography; a method of utilizing differences inan isoelectric point such as isoelectric point electrophoresis method.

When the thus obtained protein is produced as a free form, it can beconverted into a salt by the known method or a similar method, andconversely, when the protein is obtained as a salt, it can be convertedinto a free form or other salt by the known method or a similar method.By acting an appropriate protein modifying enzyme on a protein producedby a trasnformant before purification and after purification, arbitrarymodification may be performed, or a polypeptide may be partiallyremoved. As a protein modifying enzyme, there are used trypsin,chymotrypsin, arginyl endopeptidase, protein kinase, and glycosidase.

By expressing an Otx2 protein, or by increasing an expression amount ofan Otx2 protein, differentiation into a retinal photoreceptor cell canbe induced. Specifically, for example, in an eye ball tissue-derivedcell, an embryonic stem cell, a neural stem cell or a neural precursorcell, the aforementioned cells can be differentiation-induced into aretinal photoreceptor cell by expressing an Otx2 protein, or byincreasing an expression amount of an Otx2 protein. For this reason, (a)an Otx2 protein or its partial peptide, or a salt thereof, or (b) a DNAor an RNA encoding an Otx2 protein or its partial peptide can be used asan agent for inducing differentiation into a retinal photoreceptor cell.

The “inducing differentiation into a retinal photoreceptor cell” mayoccur in vivo or ex vivo. That is, an agent for inducing differentiationinto a retinal photoreceptor cell of the present invention may beadministered to a living body to induce in vivo differentiation into aretinal photoreceptor cell. Alternatively, an agent for inducingdifferentiation into a retinal photoreceptor cell of the presentinvention can be applied ex vivo to, for example, an eye balltissue-derived cell, an embryonic stem cell, a neural stem cell or aneural precursor cell, and the aforementioned cells may bedifferentiation-induced into a retinal photoreceptor cell. Morespecifically, a DNA of the present invention is introduced into an eyeball tissue-derived cell, an embryonic stem cell, a neural stem cell, aneural precursor cell, and the resulting cell is cultured, thereby toinduce differentiation of the cell into a retinal photoreceptor cell.Upon introduction of the present DNA, other gene may be introducedjointly. Examples of other gene include a retinal specific homeoboxgene. Examples of a retinal-specific homeobox gene include a gene whichhas a specific expression pattern in eye region, and regulates theregion specific pattern formation, and a gene involved in expression ofdifferentiated character in a development process. Specific examplesinclude Crx, Chx10, Pax6, and Rax.

Preferable examples of the “eye ball tissue” include an inner layertissue of the optic cup. Such tissue may be derived from an adult, ormay be derived from an individual at an embryonic stage. Examples of the“eye ball tissue-derived cell” include a fetal neural retina, a corpusciliare cell such as a corpus ciliare pigment epithelial cell or aretinal pigment epithelial cell, a corpus ciliare epithelial cell, andan iris cell.

The eye ball tissue-derived cell can be collected, for example, bytreating a tissue isolated by an appropriate means with Dispase or EDTA,treating subsequently the tissue with trypsin to separate into a singlecell, further culturing cells in an appropriate medium to confluent, andsubjecting the obtained cell to trypsin and collagenase treatment.Herein, when culturing is performed for the collection of cells, it ispossible to use media such as a medium containing basic fibroblastgrowth factor, a medium containing epithelial cell growth factor, and amedium containing ILF (leukocyte migration inhibitory factor) can beused. Examples of the medium containing basic fibroblast growth factor(bFGF) include a serum-free medium containing bFGF, more specifically,DMEM/F12 containing N₂ supplements. The content of the bFGF in suchmedium is not less than about 10 ng/ml, preferably not less than about20 ng/ml, more preferably not less than about 40 ng/ml. Examples of N₂supplements include about 5 μg/ml of insulin, about 100 μg/ml oftransferrin, about 20 nM of progesterone, about 100 μM of culturingcells in an appropriate medium to confluent, and about 30 nM of sodiumselenate. Culturing conditions such as temperature, oxygen concentrationand carbon dioxide concentration can be appropriately set, depending onthe cells.

A neural stem cell or a neural precursor cell may be derived from an eyeball tissue-derived cell, or an embryonic stem cell, or may be derivedfrom other cell or tissue. Specific examples include a fetalretina-derived neural stem cell, an adult corpus ciliare-derived retinalstem cell, an iris-derived retinal stem cell, a brain-derived neuralstem cell, a retinal precursor cell, and an iris-derived neuralprecursor cell.

A method of inducing a neural stem cell or a neural precursor cell froman eye ball tissue-derived cell or an embryonic stem cell or other cellor tissue may be performed according to the known method. For example,examples of a method of inducing a neural stem cell or a neuralprecursor cell from an embryonic stem cell include the proceduredescribed, for example, in the reference of Kawasaki, Sasai et al.(Kawasaki, H., Sasai Y., Neuron., 2000 October; 28(1):31-40). Regardingconditions for culturing and maintenance of an embryonic stem cell, forexample, “Molecular Biology Protocol” (published by Nankodo) can bereferenced. The neural stem cell or the neural precursor cell isobtained as a neural sphere containing this in some cases, but in thepresent invention, such neural sphere may be subjected to the followingprocedure.

A method of introducing the DNA of the present invention and optionalother gene into an eye ball tissue-derived cell, an embryonic stem cell,a neural stem cell or a neural precursor cell, preferably, the neuralstem cell or the neural precursor cell is not particularly limited, butthe known method may be used, and examples of such method include amethod for introducing a gene with the use of an adenovirus vector, amethod for introducing a gene with the use of a retrovirus vector, amethod for introducing a gene with the use of an adeno-associated virus,lipofection and electroporation. From a viewpoint of an introductionefficiency, preferably, a method for introducing a gene with the use ofan adenovirus vector and a method for introducing a gene with the use ofa retrovirus vector are desirable.

A cell with a gene introduced therein is cultured under differentiationinducing conditions suitable for differentiation into a retinalphotoreceptor cell. Since differentiation inducing condition isdifferent depending on a kind of a cell with a gene introduced therein,the condition can not be set primarily and can be appropriatelyselected. For example, examples of the differentiation inducingconditions include culturing in the presence of retinoic acid and serum.Herein, examples of a medium which can be used in culturing include theaforementioned DMEM/F12 medium containing N₂ supplements. It isdesirable that an amount of the retinoic acid to be used is not lessthan about 0.1 μM, preferably not less than about 0.5 μM, and not morethan about 10 μM, preferably not more than about 5 μM. In addition, itis desirable that an amount of the serum to be used is about 1% atdifferentiation inducement. Further, conditions such as temperature,oxygen concentration during culturing, and carbon dioxide concentrationcan be appropriately set depending on a cell with a gene introducedtherein.

A retinal photoreceptor cell obtained by the aforementioneddifferentiation inducing method of the present invention can be appliedas a transplantation cell for a patient with a retinal degenerativedisease such as retinitis pigmentosa, senile macular degeneration,retinal detachment, glaucoma and retinal vessel occlusion. Thetransplantation cell may be not only a cell which has been completelydifferentiated into a retinal photoreceptor cell, but also a precursorcell before differentiation into a retinal photoreceptor cell.

An Otx2 protein or its partial peptide, or a salt thereof or a DNA or anRNA encoding an Otx2 protein or its partial peptide thereof can be usedas a medicine such as an agent for preventing, treating or suppressingprogression of retinal diseases. Examples of the “retinal diseases”include retinal vessel disorders and retinal inflammatory anddegeneration lesions derived from systemic diseases such as diabetes,hypertension, arterial sclerosis, anemia, leukemia, systemic lupuserythematosus, and connective tissue diseases such as scleroderma; andinborn error of metabolism such as Tay-Sacks disease and Vogt-Spielmeyerdisease, as well as local retinal diseases including retinal vesseldisorders such as retinopathy of prematurity, retinal vein occlusion,retinal artery occlusion and retinal periphlebitis; retinal inflammationand degeneration derived from retinal detachment and trauma; age-relatedretinal degenerative diseases such as senile disciform maculardegeneration; and congenital retinal degenerative disease. Inparticular, an agent for preventing, treating or suppressing progressionof retinal diseases of the present invention can be particularlyeffectively used in congenital retinal degenerative disease, retinitispigmentosa, macular degeneration, diabetic retinopathy, retinaldetachment, glaucoma or retinal vessel occlusion.

When the protein of the present invention is used as an agent forpreventing/treating/suppressing progression of the aforementionedretinal diseases, such protein can be formulated into a preparation by aconventional means. On the other hand, when the DNA of the presentinvention is used as an agent for preventing/treating/suppressingprogression of the retinal diseases, such DNA of the present inventionalone, or after the DNA is inserted into an appropriate vector such as aretrovirus vector, an adenovirus vector, a lentivirus vector and anadenovirus-associated virus vector, can be formulated into a preparationaccording to a conventional means. The present DNA can be administeredas it is or together with an assistant for promoting uptake with a genegun or a catheter such as a hydrogel catheter.

For example, the present protein or the present DNA can be orallyadministered as a tablet optionally coated with a sugar, a capsule, anelixir, or a microcapsule, or can be parenterally administered in theform of an injection such as a sterile solution or a suspension withwater or other pharmaceutically acceptable liquid. The preparation ofthe present invention can be prepared, for example, by admixing thepresent protein or the present DNA with the physiologically acceptableknown carrier, flavor, excipient, vehicle, antiseptic, stabilizer orbinder.

Examples of the additive which can be admixed in a tablet or a capsuleinclude binders such as gelatin, corn starch, tragacanth, and gumarabic; excipients such as crystalline cellulose; swelling agents suchas corn starch, gelatin, and alginic acid; lubricants such as magnesiumstearate; sweeteners such as sucrose, lactose and saccharin; flavorssuch as peppermint, akamono (Gaultheria adenothrix) oil, and cherry. Inthe case of a capsule, a liquid carrier such as an oil and fat can befurther contained. A sterile composition for injection can be formulatedaccording to the procedure as that of a conventional formulation such asdissolution or suspension formation of an active ingredient in anaqueous solution or an oily solution for injection. As an aqueoussolution for injection, for example, an isotonic solution containingphysiological saline, glucose and other auxiliary agent (e.g.D-sorbitol, D-mannitol, sodium chloride, etc.) are used, and anappropriate solubilizer such as an alcohol (e.g. ethanol), a polyalcohol(e.g. propylene glycol, polyethylene glycol), and a nonionic surfactant(e.g. polysorbate 80 (trade mark), HCO-50) may be used together. As anoily solution, for example, a sesame oil and a soybean oil are used, andbenzyl benzoate, and benzyl alcohol which are a solubilizer may be usedtogether. Further, the sterile composition may contain, for example, abuffer (e.g. phosphate buffer, sodium acetate buffer), a soothing agent(e.g. benzalkonium chloride, procaine hydrochloride), a stabilizer (e.g.human serum albumin, polyethylene glycol), a preservative (e.g. benzylalcohol, phenol), and an antioxidant. The sterile composition preparedis usually filled into an appropriate ample, and is provided as aninjection

Since the thus obtained preparation is safe and low toxic, it can beadministered, for example, to a mammal (e.g. human, rat, mouse, rabbit,sheep, pig, cow, cat, dog, monkey, etc.). Since a dose of the presentprotein or the present DNA varies depending on administration subjects,subject organs, symptoms, and administration routes, it can not be saidgenerally, but in the case of parenteral administration, the dose isabout 0.01 to 10 mg/kg, preferably about 0.05 to 5 mg/kg, per day.

It is preferable that the agent for preventing/treating/suppressingprogression of retinal diseases of the present invention is topicallyadministered to eyes. Examples of a dosage form of such preparation fortopical administration to eyes include eye drops, opthalmic ointments,powders, granules, tablets, capsules, and injections. In particular, itis suitable that the dosage form is in the form of eye drops (e.g.aqueous eye drops, aqueous suspension of eye drops, non-aqueous eyedrops, or non-aqueous suspension of eye drops), ophthalmic ointments andinjections. Such preparations can be prepared according to theconventional manner.

Examples of diluents for aqueous solutions or suspensions used in thepreparation of eye drops include distilled water and physiologicalsaline. In addition, examples of diluents for non-aqueous solutions orsuspensions are vegetable oil, liquid paraffin, mineral oil, propyleneglycol, and p-octyldodecanol. Further, various additives such asbuffers, isotonics, preservatives, thickeners, stabilizers,antioxidants, pH adjusting agents and chelating agents which are capableof being usually admixed in eye drops can be appropriately admixed inthe eye drops of the present invention. Preparation of such eye drops isperformed by aseptic procedure or by sterilization treatment at anappropriate stage.

The above buffer is added for the purpose of maintaining the pHconstant, for example, at about 5.0 to 8.0. For example, a boratebuffer, a citrate buffer, a tartarate buffer, a phosphate buffer, and anacetate buffer are used. These buffers are added for the purpose ofadding the buffers, that is, they are added in such a range that the pHis maintained constant, for example, within the aforementioned range.The isotonic is added for the purpose of rendering isotonic with a tear,and examples of such isotonics include saccharides such as glucose,mannitol, and sorbitol; polyhydric alcohols such as glycerin,polyethylene glycol, and propylene glycol; and salts such as sodiumchloride, and sodium citrate. These isotonics are added in such anamount that an osmotic pressure of eye drops becomes equivalent to thatof a tear. Further, as the preservative, for example, benzalkoniumchloride, parabens, and chlorobutanol are used. Examples of the abovethickener include glycerin, carboxymethylcellulose and carboxyvinylpolymer. Examples of the above stabilizers include sodium sulfite, andpropylene glycol, examples of the above antioxidant include ascorbicacid, sodium ascorbate, tocopherol, and sodium thiosulfate, examples ofthe above pH adjusting agent include hydrochloric acid, citric acid,phosphoric acid, acetic acid, tartaric acid, sodium hydroxide, potassiumhydroxide, sodium carbonate and sodium bicarbonate, and examples of theabove chelating agent include sodium edetate and sodium citrate.Further, the eye drops may be lyophilized into a form which is used bydissolution in distilled water for injection upon use.

Ophthalmic ointments can be prepared under sterile conditions by mixingan active ingredient into a normally used base for ophthalmic ointments,followed by formulation according to the conventional method. Examplesof the base for ophthalmic ointments include vaseline, zelen 50,plastibase, and Macrogol, and, further, for the purpose of enhancinghydrophilicity, a surfactant may be added. In addition, regarding theophthalmic ointments, the aforementioned additives such as preservativesmay also be admixed, if necessary.

Further, a preparation for topical opthalmic administration may beformulated into a sustained-release preparation, a DDS (drug delivery)preparation, or an intraocular implant preparation using a releasecontrolling substance which can control the release of an Otx2 proteinor its partial peptide, or a DNA encoding each of them in the eyes.Examples of the release controlling substance include the known per sepolymer, copolymer, or a mixture thereof which is synthesized bynon-catalyst dehydration polycondensation from one or more kinds ofα-hydroxycarboxylic acids (e.g. glycholic acid, lactic acid,hydroxybutyric acid, etc.), hydroxydicarboxylic acids (e.g. malic acid,etc.), and hydroxytricarboxylic acids (e.g. citric acid, etc.), as wellas biodegradable polymer substances such as poly-α-cyanoacrylic acidester, polyamino acid (e.g. poly-γ-benzyl-L-glutamic acid, etc.), andmaleic anhydride-based copolymer (e.g. styrene-maleic acid copolymer,etc.).

It is not possible to generally suggest a dose and administrationfrequency of the preparation for topical ophthalmic administration ofthe present invention, because they vary depending on administrationsubjects, symptoms, dosage forms, and therapeutic periods. Usually, inthe case of eye drops, a preparation containing 0.001 to 10.0 w/v %,preferably 0.01 to 1.0 w/v %, of the present protein or the present DNAcan be administered to an adult several times a day, preferably 1 to 6times a day, per eye, at a few drops, preferably 1 to 4 drops perapplication. In the case of an ophthalmic ointment, a preparationcontaining 0.001 to 10.0 w/w %, preferably 0.01 to 1.0 w/w % of thepresent protein or the present DNA can be applied to an adult severaltimes, preferably 1 to 6 times per day.

In the present invention, diagnosis of retinal diseases can be performedby detecting an Otx2 protein or its partial peptide, or a salt thereof(hereinafter, abbreviated as present protein in some case) in a testsolution, or measuring an amount thereof. For example, when reduction ina concentration of the present protein is detected, it can be diagnosed,for example, that there is a high possibility that a person is sufferingfrom a retinal disease, or will be suffered from a retinal disease inthe future. Since an antibody to an Otx2 protein or its partial peptide,or a salt thereof (hereinafter, abbreviated as present antibody in somecases) can specifically recognize the present protein, it can be used inthe detection and quantitation of the present protein in a testsolution. That is, the present antibody can be used as a diagnosticagent for retinal diseases. In the diagnostic agent, an antibodymolecule itself may be used, or a F(ab′)₂, Fab′ or Fab fraction of anantibody molecule may also be used. Alternatively, the present proteinin a test solution may be detected by tissue staining.

The present antibody may be a polyclonal antibody or a monoclonalantibody as far as it is an antibody capable of recognizing the presentprotein. The present antibody can be prepared by using the presentprotein as an antigen according to the known process for preparing anantibody or anti-serum.

One example of the process for preparing a monoclonal antibody to thepresent protein will be described below.

(i) First, preparation of a cell producing a monoclonal antibody will bedescribed. The present protein itself or together with a carrier or adiluent is administered to a mammal at a site where an antibody can beproduced. In order to enhance antibody producing ability uponadministration, complete Freund adjuvant or incomplete Freund adjuvantmay be administered. Administration is usually performed once every 2 to6 weeks at a total of 2 to 10 times. Examples of a mammal to be usedinclude monkey, rabbit, dog, guinea pig, mouse, rat, sheep and goat, andmouse or rat is preferably used. Upon preparation of a cell producing amonoclonal antibody, a hybridoma producing a monoclonal antibody can beprepared by selecting an individual for which an antibody titre isrecognized from a warm-blooded animal, for example, a mouse immunizedwith an antigen, collecting the spleen or lymph node 2 to 5 days afterfinal immunization, and fusing an antibody-producing cell containedtherein with a myeloma cell. Measurement of an antibody titre inantibody serum can be performed, for example, by reacting a labeledprotein described later with an anti-serum, and measuring the activityof a labeling agent bound to an antibody. Fusion procedure can beperformed according to the known method, for example, the Kohler andMilstein method (Nature, vol. 256 , p. 495 (1975)). Examples of a fusionpromoter include polyethylene glycol (PEG) and Sendai virus. Preferably,PEG is used. Examples of a myeloma cell include NS-1, P3U1 and SP2/0 ,and among of them, P3U1 is preferably used. A preferable ratio of thenumber of antibody-producing cells (spleen cell) and that of myelomacells used is about 1:1 to 20:1, and PEG (preferably, PEG 1000 to PEG6000) is added at a concentration of about 10 to 80%, and incubation iscarried out at about 20 to 40° C., preferably about 30 to 37° C. forabout 1 to 10 minutes, thereby to prepare fused cells effectively.

For screening a hybridoma producing a monoclonal antibody, variousmethods can be used, including (a) a method of adding a hybridomaculture supernatant to a solid phase (e.g. microplate) onto which anantigen such as the present protein is adsorbed directly or togetherwith a carrier, subsequently adding an anti-immunoglobulin antibody(when a cell used in the cell fusion is mouse, an anti-mouseimmunoglobulin antibody is used) or Protein A labeled with a radioactivesubstance or an enzyme, and detecting a monoclonal antibody bound to thesolid phase, and (b) a method of adding a hybridoma culture supernatantto a solid phase onto which an anti-immunoglobulin antibody or Protein Ais adsorbed, adding the present protein labeled with a radioactivesubstance or an enzyme, and detecting a monoclonal antibody bound to thesolid phase. Selection of a monoclonal antibody can be performedaccording to the known method or a similar method, and such selectioncan be performed usually on a medium for an animal cell to which HAT(hypoxanthine, aminopterin, thymidine) is added. As a medium forselection and culturing, any medium may be used as far as a hybridomacan be grown. For example, RPMI 1640 medium containing about 1 to 20%,preferably about 10 to 20% fetal bovine serum, GIT medium containingabout 1 to 10% fetal bovine serum (manufactured by Wako Pure ChemicalIndustries Co., Ltd.) and a serum-free medium for hybridoma culturing(SFM-101, manufactured by Nissui Pharmaceutical Co., Ltd.) can be used.The culturing temperature is usually about 20 to 40° C., preferablyabout 37° C. The culturing time is usually about 5 days to 3 weeks,preferably about 1 week to 2 weeks. Culturing can be usually performedunder 5% carbon dioxide. An antibody titre of a hybridoma culturingsupernatant can be measured as in the measurement of an antibody titrein the above anti-serum.

(ii) Then, a monoclonal antibody is separated and purified. Separationand purification of a monoclonal antibody can be performed according toa method of separating and purifying immunoglobulin [e.g. a salting outmethod, an alcohol precipitation method, an isoelectric precipitationmethod, an electrophoresis method, an adsorbing and desorbing methodwith ion exchanger (e.g. DEAE), an ultracentrifugation method, a gelfiltration method, and a specific purification method of collecting onlyan antibody with an antigen binding to a solid phase or an activeadsorbing agent such as Protein A or Protein G, and releasing thebinding to obtain an antibody] as in conventional separation andpurification of a polyclonal antibody.

One example of a process for preparing a polyclonal antibody to thepresent protein (hereinafter, abbreviated as “Present polyclonalantibody” in some cases) will be described below.

The preset polyclonal antibody can be prepared according to the knownmethod or a similar method thereof. For example, the antibody can beprepared by making a complex of an immunological antigen (presentprotein, etc.) and a carrier protein, immunizing a mammal as in theaforementioned process for preparing a monoclonal antibody, collectingan antibody containing composition against the present protein from theimmunized animal, and separating and purifying an antibody. Regarding acomplex of an immunological antigen with a carrier protein used forimmunizing a mammal, the kind of a carrier protein, and the ratio ofmixing a carrier and a hapten is not particularly limited as far as anantibody can be effectively produced against a hapten immunized bycrosslinking with a carrier. For example, a method of coupling bovineserum albumin, bovine thyroglobulin, keyhole lympet hemocyanin at aweight ratio of about 0.1 to 20 , preferably about 1 to 5 relative to ahapten is used. In addition, for coupling a hapten with a carrier,various condensing agents can be used, including glutaraldehyde, acarbodiimide, a maleimide active ester, and an active ester reagentcontaining a thiol group or a dithiopyridyl group are used. Acondensation product from a hapten and a carrier itself or together witha carrier and a diluent is administered to a warm-blooded animal at asite where an antibody can be produced. In order to enhance antibodyproductivity upon administration, complete Freund adjuvant or incompleteFreund adjuvant may be administered. Administration can be usuallyperformed once per about 2 to 6 weeks at a total of about 3 to 10 times.A polyclonal antibody can be collected from blood or ascites, preferablyblood of a mammal immunized by the aforementioned method. A polyclonalantibody titre in an anti-serum can be measured as in the aforementionedmeasurement of an antibody titre in anti-serum. Separation andpurification of a polyclonal antibody can be performed according to asimilar method of separating and purifying immunoglobulin to theaforementioned separation and purification of a monoclonal antibody.

A method of quantitating the present protein using an antibody of thepresent invention is not particularly limited, but examples of suchmethods include a method of detecting an amount of an antibody, anantigen or an antibody-antigen complex corresponding to an amount of anantigen (amount of the present protein, etc.) in a test solution by achemical or physical means, and calculating an amount of an antigen in atest solution based on a standard curve produced with the use of astandard solution containing the known amount of an antigen from adetected value. For example, nephrometry, a competition method, animmunometric method and a sandwich method are preferably used, but froma viewpoint of sensitivity and specificity, it is particularlypreferable to use a sandwich method described later. A labeling agentused in a quantitation method using a labeling substance includes, forexample, a radioactive isotope element, an enzyme, a fluorescentsubstance, and a light emitting substance. As the radioactive isotopeelement, for example (¹²⁵I), (¹³¹I), (³H) and (¹⁴C) are used. As theenzyme, those which are stable and have large specific activity arepreferable, and for example, β-galactosidase, β-glucosidase, alkalinephosphatase, peroxidase, and malate dehydrogenase are used. As thefluorescent substance, for example, fluorescamine, and fluoresceinisothiocyanate are used. As the light emitting substance, luminol,luminol derivative, luciferin and lucigenin are used. Further,biotin-avidin system may be used for binding an antibody or an antigenwith a labeling agent.

When an antigen or an antibody is insolubilized in the aforementionedquantitation method, physical adsorption may be used, or chemicalbinding which is usually used for insolubilizing/immobilizing a proteinor an enzyme may be used. As a carrier, for example, insolublepolysaccharides such as agarose, dextran and cellulose; synthetic resinssuch as polystyrene, polyacrylamine and silicone; and glass are used. Ina sandwich method, an amount of the present protein in a test solutioncan be quantitated by reacting the insolubilized present monoclonalantibody with a test solution (primary reaction), further reacting thelabeled present monoclonal antibody (secondary reaction), and measuringthe activity of a labeling agent on an insolubilized carrier. Theprimary reaction and the secondary reaction may be performed in areverse order, or may be performed at the same time. The labeling agentand the method of insolubilization are the same as described above. Inaddition, in an immunological quantitation method by a sandwich method,an antibody used in a solid phase antibody or a labeling antibody is notnecessarily one kind, and a mixture of two or more kinds of antibodiesmay be used for the purpose of improving measurement sensitivity. In amethod of quantitating the present protein by a sandwich method, as thepresent monoclonal antibodies used in a primary reaction and a secondaryreaction, it is preferable to use antibodies having different sites forbinding with the present protein. That is, antibodies used in a primaryreaction and a secondary reaction are such that, for example, when anantibody used in a secondary reaction recognizes a C-terminal part ofthe present protein, an antibody recognizing a part other than theC-terminal part, for example, an N-terminal part, used in a primaryreaction, is preferably used.

The present monoclonal antibody can also be used in a quantitationmethod other than a sandwich method, such as a competition method, animmunometric method and nephrometry. In the competition method, anantigen and a labeled antigen in a test solution are reactedcompetitively to an antibody, an unreacted labeled antigen (F) and alabeled antigen (B) bound with an antibody are separated (B/Fseparation), a label amount of either B or F is measured, and an amountof an antigen in a test solution is quantitated. Specifically, examplesof the present method include (a) a liquid phase method wherein asoluble antibody is used as an antibody, and B/F separation is performedusing polyethylene glycol and a second antibody to the antibody, and (b)a solid phase method using a solid phased antibody as a first antibody,or using a soluble antibody as a first antibody, and a solid phasedantibody as a second antibody. In the immunometric method, after anantigen in a test solution and a solid phased antigen are reactedcompetitively against a constant amount of a labeled antibody, the solidphase and the liquid phase are separated, or an antigen in a testsolution and an excessive amount of a labeled antibody are reacted,then, a solid phased antigen is added to bind an unreacted labeledantibody to a solid phase, and the solid phase and the liquid phase areseparated. Then, the amount of a labeled antibody in any of phases ismeasured to quantitate the amount of an antigen in a test solution. Inaddition, in nephrometry, an amount of an insoluble precipitate producedas a result of an antigen antibody reaction in a gel or in a solution ismeasured. When an amount of an antigen in a test solution is small, andonly a small amount of precipitate is obtained, laser nephrometryutilizing laser light scattering is preferably used.

When these individual immunological quantitation methods are applied tothe present invention, normal technical consideration of a personskilled in the art may be added to an operation method according toconventional conditions in each method. For details of these generaltechnical means, review, and books can be referenced (e.g. see“Radioimmunoassay” edited by Hiroshi Irie (Kodansha, published in 1974);Separate Volume ‘Radioimmunoassay” edited by Hiroshi Irie (Kodansha,published in 1979); “Enzyme Immunoassay” edited by Eiji Ishikawa et al.(Igakushoin, published in 1978); “Enzyme Immunoassay” edited by EijiIshikawa (second edition) (Igakushoin, published in 1982); “Enzymeimmunoassay” edited by Eiji Ishikawa et al. (third edition) (Igakushoin,published in 1987); “Methods in ENZYMOLOGY”, Vol. 70 (ImmunochemicalTechniques (Part A)); ibid Vol. 73 (Immunochemical Techniques (Part B));ibid Vol. 74 (Immunochemical Techniques (Part C)); ibid Vol. 84(Immunochemical Techniques (Part D: Selected Immunoassays); ibid Vol. 92(Immunochemical Techniques (Part E: Monoclonal antibodies and GeneralImmunoassay Methods)); ibid Vol. 121 (Immunochemical Techniques (Part I:Hybridoma Technology and Monoclonal Antibodies)) (all published byAcademic Press).

The present antibody can also be used for preparing an antibody columnused for purifying the present protein, detecting the present protein ineach fraction at purification, or analyzing behavior of the presentprotein in a test cell.

Since the present DNA, or an antisense polynucleotide comprising anucleotide sequence which is complementary or substantiallycomplementary to a nucleotide sequence of the present DNA can be used asa probe, and thus can detect abnormality (gene abnormality) of a DNA ora mRNA encoding the present protein or its partial peptide in a livingbody, in particular, in a living body of a mammal (e.g. human, rat,mouse, rabbit, sheep, pig, cow, cat, dog, monkey, etc.), they areuseful, for example, as an agent for genetic diagnosis of damages of theDNA or the mRNA, or mutation, or reduction in expression. The geneticdiagnosis using the present DNA or antisense polynucleotide can beperformed, for example, by the known Northern hybridization or PCR-SSCPmethod (Genomics, vol. 5 , p. 874-879 (1989), Proceedings of theNational Academy of Sciences of the USA, vol. 86, p. 2766-2770 (1989)).For example, when reduction in expression of a mRNA encoding the presentprotein or its partial peptide is detected by Northern hybridization, itcan be diagnosed that there is a high possibility that a subject issuffering from a retinal disease, or will be suffered from a retinaldisease in the future.

It is enough that the “antisense polynucleotide” is a polynucleotidehaving a nucleotide sequence complementary to a nucleotide sequence ofthe present DNA in at least a part, and capable of hybridizing thepresent DNA. Therefore, antisense polynucleotide may be not only anentity comprising a nucleotide sequence complementary to a nucleotidesequence of the present DNA, but also an entity comprising a nucleotidesequence substantially complementary to a nucleotide sequence of thepresent DNA. Examples of the antisense polynucleotide include an entitycomprising a nucleotide sequence which hybridizes with the present DNAunder highly stringent conditions. Examples of the antisensepolynucleotide include other type polynucleotides other than theaforementioned polynucleotides, such as a polydeoxynucleotide containing2-deoxy-D-ribose, a polynucleotide containing D-ribose, and an entitycontaining N-glycoside of a purine or pyrimidine base; other polymershaving a non-nucleotide skeleton (e.g. commercially available proteinnucleic acid, and polymer of nucleic acid having specific syntheticsequence); and other polymers having a special bond (provided that thepolymer contains a nucleotide having arrangement permitting pairing ofbases or addition of bases found in a DNA or an RNA). These may be adouble-stranded DNA, a single-stranded DNA, a double-stranded RNA, asingle-stranded RNA, or a DNA/RNA hybrid, and may be a non-modifiedpolynucleotide (or non-modified oligonucleotide), or a known modifiedpolynucleotide (or modified oligonucleotide) to which a modification isadded. Examples of a modified polynucleotide include a polynucleotidehaving a label known in the art, a polynucleotide with a cap, amethylated polynucleotide, a polynucleotide in which one or more naturalnucleotides are substituted with analogs, an intramolecularnucleotide-modified polynucleotide, a polynucleotide having a non-chargebond (e.g. methylphosphonate, phosphotriester, phosphoramidate,carbamate, etc.), a polynucleotide with a bond having a charge or asulfur-containing bond (e.g. phosphorothioate, phosphorodithioate), apolynucleotide having, as aside group, a protein (nuclease, nucleaseinhibitor, toxin, antibody, signal peptide, poly-L-lysine) or asaccharide (e.g. monosaccharide), a polynucleotide having an intercalentcompound (e.g. acridine, solarene, etc.), a polynucleotide containing achelating compound (e.g. metal, radioactive metal, boron, oxidativemetal, etc.), a polynucleotide containing an alkylating agent, and apolynucleotide having a modified bond (e.g. α-anomer type nucleic acid).Herein, “nucleoside”, “nucleotide” and “nucleic acid” may contain notonly a purine and pyrimidine base, but also other modified heterocyclicbase. These modified entities may contain methylated purine andmethylated pyrimidine, acylated purine and acylated pyrimidine or otherheterocycles. A modified polynucleotide having a saccharide as a sidechain group may be such that a sugar part of a side chain is furthermodified, for example, one or more hydroxyl groups of a saccharide aresubstituted with a halogen or an aliphatic group, or are converted intoa functional group such as ether and amine.

That is, the antisense polynucleotide of the present invention is anRNA, a DNA, or a modified nucleic acid (RNA, DNA). Examples of themodified nucleic acid include a sulfur derivative and a thiophosphatederivative of nucleic acid, and nucleic acid which is resistant todegradation of polynucleotide amide or oligonucleoside amide, which isnot limited thereto.

Using expression or a rise in an expression amount of an Otx2 protein orits partial peptide as an index, a compound having action of inducingdifferentiation into a retinal photoreceptor cell or a salt thereof canbe screened. The screening can be performed, for example, using a cellhaving an ability to express an Otx2 protein or its partial peptide.

Specifically, examples of such screening include a method for screeninga compound having an action of inducing differentiation into a retinalphotoreceptor cell or a salt thereof, characterized in that a cellhaving an ability to express an Otx2 protein or its partial peptide iscultured in the presence of a test compound, and expression of an Otx2protein or a partial peptide thereof is detected, or an expressionamount of them is measured. Examples of the “cell having an ability toexpress an Otx2 protein or its partial peptide” include a transformantcell having the aforementioned present DNA. Alternatively, the cell maybe a cell originally having an ability to express an Otx2 protein or itspartial peptide not based on gene recombinant technique. An amount ofexpression of the present protein can be measured using theaforementioned method of quantitating the present protein by separatingand purifying the present protein from a cultured cell by theaforementioned method.

In addition, other aspect of the present screening method includes amethod for screening a compound having an action of inducingdifferentiation into a retinal photoreceptor cell or a slat thereof,characterized in that a cell having an ability to express an Otx2protein or its partial peptide thereof is cultured in the presence of atest compound, and an amount of a mRNA encoding an Otx2 protein(hereinafter, abbreviated as Otx2 mRNA in some cases) is measured usinga DNA encoding the present protein or a complementary DNA thereof or apartial DNA thereof. More specifically, there is provided a method forscreening a compound having an action of inducing differentiation into aretinal photoreceptor cell or a salt thereof, characterized in thatcomparison was made between (a) an amount of expression of an Otx2 mRNAwhen a cell having an ability to express an Otx2 protein or its partialpeptide is cultured, and (b) an amount of an Otx2m RNA when a cellhaving an ability to express an Otx2 protein or its partial peptide iscultured in the presence of a test compound.

In order to perform comparison of expression amounts of a mRNA by ahybridization method, such comparison can be performed according to theknown method or a similar method thereof, for example, the methoddescribed in Molecular Cloning, 2^(nd), J. Sambrook et al., Cold SpringHarbor Lab. Press, 1989. Specifically, measurement of an amount of amRNA encoding an Otx2 protein is performed by contacting a mRNAextracted from a cell according to the known method with a DNA encodingthe present protein or a complementary DNA thereof or a partial DNAthereof, and measuring an amount of a mRNA bound to a DNA encoding thepresent protein or a complementary DNA thereof or a partial DNA thereof.An amount of an Otx2 mRNA bound to a DNA encoding the present protein ora complementary DNA thereof or a partial DNA thereof can be easilymeasured by labeling a DNA encoding the present protein or itscomplementary DNA or a partial DNA thereof with, for example, aradioactive isotope element or a pigment. As the radioactive isotopeelement, for example, ³²P and ³H are used and, as a pigment, fluorescentpigments such as fluorescein, FAM (manufactured by PE Biosystems), JOE(manufactured by PE Biosystems), TAMRA (manufactured by PE Biosystems),ROX (manufactured by PE Biosystems), Cy5 (manufactured by Amershan) andCy3 (manufactured by Amershan) are used. Alternatively, an amount of anOtx2m RNA can also be measured by converting a RNA extracted from a cellinto a cDNA with a reverse transcriptase, and measuring an amount of acDNA which has been amplified by PCR using, as a primer, a DNA encodingthe present protein or its complementary DNA or a partial DNA thereof.

There is provided a method for screening a compound having an action ofcontrolling activity of a promoter or an enhancer of a DNA encoding anOtx2 protein or a salt thereof, further a method for screening acompound having an action of inducing differentiation into a retinalphotoreceptor cell or a salt thereof, characterized in that the knownpromoter or enhancer region of the DNA encoding an Otx2 protein iscloned from a genome DNA, a cell transformed with a recombinant DNAligated upstream of an appropriate reporter gene cultured in thepresence of a test compound, and expression of a reporter gene in placeof expression of an Otx2 protein is detected. As a reporter gene, astaining marker gene such as lacZ (β-galactosidase gene) is used. Bymeasuring an amount of a reporter gene product (e.g. mRNA, protein)using the known method, a test compound of increasing an amount of areporter gene product can be selected as a compound having an action ofpromoting activity of a promoter or an enhancer of an Otx2 gene, thatis, a compound having an activity of promoting expression of an Otx2protein or its partial peptide.

Examples of a test compound in the aforementioned present screeningmethod include a peptide, a protein, a non-peptidic compound, asynthetic compound, a fermentation product, a cell extract, a plantextract, and an animal tissue extract, and these compounds may be anovel compound, or the known compound.

In order to perform the aforementioned screening method, the presentscreening kit contains (a) a cell having an ability to express an Otx2protein or its partial peptide, (b) a DNA encoding the present proteinor a complementary DNA thereof or a partial DNA thereof, or (c) a celltransformed with a DNA in which a promoter or an enhancer of a DNAencoding an Otx2 protein is ligated to a reporter gene.

A compound obtained using the present screening method or screening kit,or a salt thereof is useful as a drug such as the aforementioned agentfor preventing, treating or suppressing progression of a retinaldisease, and an agent for inducing differentiation into a retinalphotoreceptor cell. The compound or a salt thereof, an agent forpreventing, treating or suppressing progression of a retinal diseasecontaining it, or an agent for inducing differentiation into a retinalphotoreceptor cell containing it can be performed as in the presentprotein or DNA.

EXAMPLES

As shown in FIG. 1, a mouse Otx2 cDNA was incorporated into a LIA vectorwhich is a retrovirus vector. A human placenta-derived alkalinephosphatase gene is incorporated into a LIA vector (control virusvector), and a cell infected with a virus derived from this vectorexpresses alkaline phosphatase as a marker. A cell infected with a virusderived from a LIA vector with an Otx2 gene incorporated therein (Otx2virus vector) expresses an Otx2 protein and, at the same time,coexpresses alkaline phosphatase as a marker.

Using a cultured cell (Phoenix cell line) for producing a retrovirus, acontrol virus vector and an Otx2 virus vector were prepared,respectively, the resulting viruses were concentrated byultracentrifugation (swing rotor, 21,000 rpm 4° C. for 2 hours) toprepare a virus solution having an infection efficiency of 1×10⁷ pfu(plaque forming unit)/ml, respectively.

Then, as shown in FIG. 2, an infant rat immediately after birth (0 dayafter birth) was subjected to low temperature anesthesia, and a skincovering an ocular part was excised with operation scissors. 5 μl of thevirus solution of a control virus vector or an Otx2 virus vector wasinjected under a retina of an infant rat using an injection needle(manufactured by Hamilton). After injection, an infant rat was warmed at37° C. for 20 minutes to recover a body temperature, returned to abreeding cage of a mother rat, and subsequently reared for 4 to 6 weeks.

A rat grown to adult was subjected to euthanasia by administering sodiumpentobarbital. Eyes were isolated from the rat, and retina was removed.The retina was fixed overnight at 4° C. with a 4% paraformaldehydesolution. A 4% paraformaldehyde solution was exchanged with PBS(Phosphate Buffer Saline), and a fixed retina was washed. Thisfixation/washing operation was repeated three times. Then, retina fixedwith a 4% paraformaldehyde solution was thermally treated at 65° C., andendogenous alkaline phosphatase was inactivated. Thermally treatedretina was stained with an alkaline phosphate staining solution (roomtemperature, 3 hours) to stain only a retinal cell infected with theaforementioned virus in blue purple. This stained retina was fixed againovernight with a 4% paraformaldehyde solution, washed with PBS, then,immersed overnight in 30% sucrose/PBS solution overnight, transferredinto an OTS compound (Sakura Finetek) liquid, and a frozen block ofretina was prepared on dry ice. An about 30 μm frozen section wasprepared from a frozen block using a frozen section preparing apparatus(Karl Zeis), and this was observed under the optical microscope (KarlZeis).

A kind of each cell of retina was identified by its form and position(see FIG. 3). This was performed three times by an independent test. Anexistence rate of each cell relative to all cell number seen in onefield is shown in FIG. 4. When an Otx2 virus vector was introduced intoa retinal stem cell (or retinal precursor cell), the number of retinalphotoreceptor cells was increased by about 10% as compared with the casewhere a control virus vector was introduced. From this, it was seenthat, by expression of an Otx2 gene, differentiation from a retinal stemcell into a bipolar cell, an amacrine cell and a Muller glian cell wasstrongly suppressed, and almost of retinal stem cells are differentiatedinto retinal photoreceptor cells.

From the above result, it was confirmed that, by introducing an Otx2gene into an undifferentiated retinal stem cell, and expressing an Otx2gene in the cell, it is possible to effectively differentiate anundifferentiated retinal stem cell of a rat into a retinal photoreceptorcell.

INDUSTRIAL APPLICABILITY

The present protein or the present DNA can be utilized in a drug for thepurpose of preventing, treating or suppressing progression of retinaldiseases such as retinitis pigmentosa, senile macular degeneration,diabetic retinopathy, retinal detachment, glaucoma and retinal vesselocclusion. Further, since abnormality of an Otx2 gene leads tostructural or functional abnormality of a retinal photoreceptor cell, bydetecting abnormality of an Otx2 gene or degeneration or reduction inexpression of an Otx2 protein, this can be utilized in diagnosis of theaforementioned disease.

1. A method for inducing differentiation into a retinal photoreceptorcell and suppressing differentiation into a bipolar cell, an amacrinecell and a Muller glia cell, which method comprises: introducing a DNAor an RNA encoding an Otx2 protein into an eye ball tissue-derived cell,an embryonic stem cell, a neural stem cell or a neural precursor cell;and culturing the resulting cell containing the DNA or RNA encoding anOtx2 protein to express an Otx2 protein, or increase an amount ofexpression of an Otx2 protein, wherein the Otx2 protein is a proteinhaving the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ IDNO:5.
 2. The method according to claim 1, which method comprises:introducing a DNA or an RNA encoding an Otx2 protein into an eye balltissue-derived cell or an embryonic stem cell; and culturing theresulting cell containing the DNA or RNA encoding an Otx2 protein toexpress an Otx2 protein, or increase an amount of expression of an Otx2protein, wherein the Otx2 protein is a protein having the amino acidsequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.
 3. The methodaccording to claim 2, wherein the eye ball tissue-derived cell is a cellselected from the group consisting of a retinal cell, a retinal stemcell, a retinal precursor cell, a corpus ciliare cell, a corpus ciliareepithelial cell, a retinal pigment epithelial cell and an iris cell. 4.The method according to claim 1, which method comprises: introducing aDNA or an RNA encoding an Otx2 protein and a nucleic acid encoding aretinal specific homeobox protein selected from the group consisting ofCrx, Chx10, Pax6 and Rax into an eye ball tissue- derived cell, anembryonic stem cell, a neural stem cell or a neural precursor cell; andculturing the resulting cell containing the DNA or RNA encoding an Otx2protein to express an Otx2 protein, or increase an amount of expressionof an Otx2 protein, wherein the Otx2 protein is a protein having theamino acid sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.
 5. Themethod according to claim 1, which method comprises: introducing a DNAor an RNA encoding an Otx2 protein into an eye ball tissue-derived cell,an embryonic stem cell, a neural stem cell or a neural precursor cellusing an adenovirus vector, a retrovirus vector, an adeno-associatedvirus vector, lipofection or electroporation; and culturing theresulting cell containing the DNA or RNA encoding an Otx2 protein toexpress an Otx2 protein, or increase an amount of expression of an Otx2protein, wherein the Otx2 protein is a protein having the amino acidsequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.
 6. The methodaccording to claim 1, which method comprises: introducing a DNA or anRNA encoding an Otx2 protein into an eye ball tissue-derived cell, anembryonic stem cell, a neural stem cell or a neural precursor cell; andculturing the resulting cell containing the DNA or RNA encoding an Otx2protein in the presence of retinoic acid and serum to express an Otx2protein, or increase an amount of expression of an Otx2 protein, whereinthe Otx2 protein is a protein having the amino acid sequence of SEQ IDNO:1, SEQ ID NO:3 or SEQ ID NO:5.